Feruloyl esterase Fae1 is required specifically for host colonisation by the rice-blast fungus Magnaporthe oryzae

Plant cell wall acts as a primary barrier for microbial pathogens during infection. A cell wall-degrading enzyme thus may be a crucial virulence factor, as it may aid the pathogen in successful host invasion. Nine genes coding for feruloyl esterases (Fae), likely involved in plant cell wall degradation, have been annotated in the genome of the cereal-blast fungus Magnaporthe oryzae. However, role of any Fae in pathogenicity of M. oryzae remains hitherto under explored. Here, we identified FAE1 gene (MGG_08737) that was significantly upregulated during host penetration and subsequent colonisation stages of infection. Accordingly, while deletion of FAE1 in M. oryzae did not affect the vegetative growth and asexual development, the fae1Δ mutant showed significantly reduced pathogenesis on rice plants, mainly due to impaired host invasion and colonisation. Very few (< 10%) fae1Δ appressoria that formed the primary invasive hyphae failed to elaborate from the first invaded cell to the neighbouring plant cells. Interestingly, exogenously added glucose, as a simple carbon source, or ferulic acid, a product of the Fae activity, significantly supported the invasive growth of the fae1Δ mutant. We show that the Fae1-based feruloyl esterase activity, by targeting the plant cell wall, plays an important role in accumulating ferulic acid and/or sugar molecules, as a likely energy source, to enable host invasion and colonisation by M. oryzae. Given its role in plant cell wall digestion and host colonisation, M. oryzae Fae1 could be a potential candidate for a novel antifungal strategy and a biotechnological application in biofuel production.


Introduction
Microbial phytopathogens encounter plant cell wall as a major obstruction while invading and successfully colonising the host. Typical plant cell wall is primarily composed of three polysaccharides, namely, cellulose (microfibrils), hemicellulose (xylan and xylan derivatives), and pectin, interconnected via ferulic acid bridges to form a rigid meshlike structure (Harris and Hartley, 1977;Bunzel et al. 2001). This mesh-like network is required for providing strength and integrity to the plant cell wall. Phytopathogens, such as fungi and bacteria, deploy different ways to overcome this physical cell wall barrier, mainly to get entry into the host cell and colonise the tissue for nutrient acquisition. While bacterial pathogens prefer passive host entry via hydathodes or stomatal openings, fungal pathogens have evolved mechanisms to breach the primary barrier of plant hosts. The necrotrophic fungal pathogens use cell wall-degrading enzymes (CWDE) to invade/colonise the plant tissue (Cosgrove 2001), whereas the biotrophic or hemibiotrophic fungal pathogens, in addition to CWDE, depend on specialised host entry enabled by infection structures called as appressorium (Howard et al. 1991). While, CWDEs are fairly studied with respect to virulence of phytopathogenic fungi, they are also implemented in industrial applications such as processing of plant cell walls for efficient production of biofuels. Ever-increasing collection of genome sequences reveals that CWDEs offer a wide diversity, both in terms of number and expansion of gene families, across phytopathogenic fungi (Kubicek et al. 2014). It is therefore important to study different CWDEs, which may lead to identification of novel virulence determinants in different phytopathogenic fungi.
Feruloyl esterases (ferulic acid esterases, Fae; EC 3.1.1.73), a subclass of carboxylic acid esterases (EC 3.1.1.1), also belong to one such group of CWDEs. The hydrolyses the ester bonds in the feruloyl-polysaccharide complex in the plant cell wall and thereby releases ferulic acid and polysaccharide (Faulds and Williamson 1994;de Vries et al. 1997). Breakage of these ester bonds leads to loss of elasticity and plasticity and subsequent weakening of the plant cell wall. Feruloyl esterases have been classified into four classes-type A, B, C, and D-depending upon sequence attributes and ability to act on wide range of substrates (Crepin et al. 2004). Crystal structure of feruloyl esterase from Aspergillus niger (AnFaeA) has revealed that it is a modular enzyme with a catalytic and a non-catalytic cellulose-binding domain (Hermoso et al. 2004). Involvement of fungal feruloyl esterases with differential activity during plant infection has been studied in different pathosystems. In Fusarium graminearum, feruloyl esterases, particularly FaeB1 and FaeD1, are found to be upregulated during infection or in response to aromatic compounds such as ferulic acid, caffeic acid, and p-coumaric acid, and also in the presence of carbon sources such as xylose, glucose, and galactose (Balcerzak et al. 2012). However, FaeB1 and FaeD1 are not required for pathogenicity on wheat (Balcerzak et al. 2012). On the other hand, expression of Fae is not only upregulated during infection but also plays an essential role in pathogenesis in the apple tree canker pathogen Valsa mali (Xu et al. 2017).
Magnaporthe oryzae (synonym Pyricularia oryzae), a hemibiotrophic phytopathogenic filamentous fungus, causes blast disease in rice and other important cereal crops worldwide (Valent and Khang 2010). Over several years, riceblast disease has been widely used as a model pathosystem to study plant-pathogen interactions (Ebbole 2007;Patkar et al. 2015). The infection cycle of M. oryzae starts with conidial germination in the presence of moisture, followed by perception of specific cues from the host surface, leading to development of appressorium. The enormous turgor pressure generated inside the appressorium helps the fungus in penetrating the host cell. While the intracellular turgor pressure inside the appressorium contributes in generating the mechanical force, the localised loosening of the host cell wall underneath the appressorium is important and carried out by certain plant cell wall digesting enzymes secreted by the fungal pathogen. During subsequent host tissue invasion, the penetration peg differentiates into bulbous primary invasive hypha that elaborates within the first invaded cell. Once inside the first host cell, the fungal pathogen uses different strategies to evade plant immunity to colonise the tissue and for disease progression (Skamnioti and Gurr 2008;Patkar et al. 2015).
Role of CWDEs in host penetration and thereby virulence has been studied in M. oryzae. Endo-xylanases and cellulases are significantly upregulated during plant infection and required for penetration and virulence in M. oryzae (Nguyen et al. 2011;Vu et al. 2012). Furthermore, a secreted feruloyl esterase, encoded by MGG_01403.5, in M. oryzae is found to be expressed during post-penetration stage (72 hpi onwards) of rice infection, but does not play a significant role in pathogenesis of the fungus (Zheng et al. 2009). Interestingly, the FAE gene family in M. oryzae is relatively expanded when compared to that in non-pathogenic counterparts such as Neurospora crassa and Aspergillus nidulans, which have only one and three FAE genes, respectively (Dean et al. 2005). Hitherto, role of any other FAEs, particularly type B feruloyl esterases, has not been studied in M. oryzae. Here, we identified, through in silico analysis, nine putative type B Fae sequences in M. oryzae. We studied the expression profiles of these nine FAE genes, to identify an early invasion-related Fae function. We show a crucial role for one of the Fae, specifically in host invasion and tissue colonisation.

Experimental procedures
Fungal culture and growth conditions M. oryzae wild-type (WT) B157 strain (MTCC accession no. 12236;Kachroo et al. 1994) belonging to the international race IC9 was used in this study. Fungus was grown and maintained on Prune Agar (PA) plates as described earlier (Soundararajan et al. 2004). Vegetative growth of the fungus on PA plates was allowed for 10 days at 28 °C, with initial 3 day incubation under dark conditions followed by 7 day incubation under constant illumination for conidiation. Vegetative growth was assessed by visual observation of the colony morphology and by measuring the colony diameter. Conidia were harvested as described previously (Patkar et al. 2010), followed by microscopic observation of the conidial morphology. Harvested conidia were counted using a hemocytometer and reported in terms of total number of conidia per unit area of the colony.
Assay for appressorial development was performed by spotting 20 µL conidial suspension (~ 10 4 conidia/mL) on an inductive (hydrophobic) cover glass (22 mm, no. 1; Microcil Ltd., India) for up to 24 h at 25 °C under humid conditions, followed by assessment of appressorium formation by microscopic observation.
Nucleic acids and proteins were isolated by grinding in liquid nitrogen the fungal biomass obtained from vegetative culture grown in an appropriate liquid medium for 2-3 days at 28 °C, followed by the standard protocols mentioned earlier (Dellaporta et al. 1983;Kachroo et al. 1997).

Identification and in silico analysis of fungal Fae
Initial identification of putative feruloyl esterases in M. oryzae was done using NCBI protein BLAST (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi? PAGE= Prote ins) with known type B Fae sequences from Aspergillus oryzae (AoFaeB; PDB: 3WMT_B) and Neurospora crassa (NcFaeB; Gen-Bank: AJ293029). Multiple sequence alignment of these putative Fae was carried out using ClustalW feature in MEGA tool to check the presence of GXSXG conserved motif (Dilokpimol et al. 2016). Presence of the characteristic α/β hydrolase domain was checked using the NCBI conserved domain database (http:// www. ncbi. nlm. nih. gov/ Struc ture/ cdd/ wrpsb. cgi; Marchler-Bauer et al. 2015). Furthermore, percentage identity among all these M. oryzae Fae was checked by performing multiple sequence alignment of protein sequences using Clustal Omega (Sievers et al. 2011;Sievers and Higgins, 2018), followed by plotting the distance matrix heatmap using R tool (Supplementary Fig. S6).
Phylogenetic analysis of Fae in different host-specific isolates of M. oryzae (GY11, P131, Y34, PH14, US71, MZ5-1-6, CD156, and BR32) was carried out using the annotated protein sequences, available in the NCBI and GEMO (http:// genome. jouy. inra. fr/ gemo/) databases, from these isolates. Protein sequences of putative Fae in M. oryzae 70-15 strain were used as a query to perform BlastP analysis with a custom database containing all the protein sequences from the aforementioned different isolates. Phylogenetic analyses were carried out with MEGA11 (Tamura et al. 2021), using the maximum-likelihood method based on the JTT matrix-based model.
For phylogenetic analysis of feruloyl esterases across different fungal taxa, genome sequences of 26 representative fungal species (Supplementary Table S1 and Supplementary Information_2) were retrieved from the NCBI database. Proteomes were mined for the presence of the conserved domain Tannase (Pfam ID: PF07519.13) or Esterase_PHB (Pfam ID: PF10503.13), using hmmsearch (hidden Markov model search) of HMMER suite ver. 3.3.2 (parameters -cut_tc; Eddy 2011). Poorly aligned sequences were removed using TrimAl (Capella-Gutierrez et al. 2009;parameters -automated1). The Tannase domain containing protein sequences were curated manually and were aligned to construct the maximum-likelihood phylogenetic tree, using IQ-TREE ver. 2.1.2 (parameters -m MFP -alrt 1000 -bb 1000 -nt AUTO; Minh et al. 2020). Best-fit model (LG + R6) was chosen based on the Bayesian Information criterion (BIC), followed by assessment of phylogenetic tree for branch support with SH-like approximate likelihood ratio test (alrt) and ultrafast bootstrap (bb). Phylogenetic tree was visualized using iTOL (Letunic and Bork 2021).

Signal peptide prediction and validation
Prediction of conventional secretory signal peptide was carried out using SignalP 4.0 tool (http:// www. cbs. dtu. dk/ servi ces/ Signa lP-4. 0/; Petersen et al. 2011). Presence of functional signal peptide was confirmed for two randomly selected FAEs-MGG_05529 and MGG_07294-using Yeast Secretion Trap (YST) approach as described (Lee et al. 2006). The ORFs of MGG_05529 and MGG_07294 were PCR-amplified from M. oryzae WT genomic DNA, using the following primer pairs (MGG_05529-F: 5′-ATG GAC TCG TCA ATC ATT CAC TGG -3′ and MGG_05529-R: 5′-CCC CAT TCC ACT TTG ACC TG-3′; MGG_07294-F: 5′-ATG CGT TTC TCC AGC ATC TTC-3′ and MGG_07294-R: 5′-CGC AAT GAG ACC AAA GAA CC-3′). Individual PCR products were subjected to blunt end cloning in pYST2 vector at NotI site after end-filling, followed by E. coli DH5α transformation. Transformants obtained on Luria-agar plates containing 100 µg/mL ampicillin antibiotic were screened by RE digestion and those with desired restriction digestion pattern were confirmed by DNA sequencing. These recombinant plasmids from desired clones of E. coli DH5α were used for yeast (Saccharomyces cerevisiae) DBYα2445 (MATα, transformation as described previously (Gietz and Woods 2002). Selected transformants were screened and confirmed by colony PCR. Confirmed S. cerevisiae transformants were spotted on SD (YNB with (NH 4 ) 2 SO 4 , Lysine, Uracil, and Adenine) + sucrose agar selection plates and incubated for 6 days at 28 °C.

Determination of fungal Fae activity
To check the effect of host extract on Fae enzyme activity, 3-day-old vegetative culture of WT M. oryzae grown in liquid YEG (0.2% yeast extract and 1% glucose) medium with or without crude rice leaf extract was used. Protein samples were prepared from fungal biomass (intracellular proteins) and culture supernatant (secretory proteins), essentially as described earlier (Kachroo et al. 1997), followed by biochemical spectrophotometric enzyme assay using Fae-specific substrate, 4-nitrophenyl ferulate (Institute of Chemistry, Slovak Academy of Sciences, Bratislava), as described previously (Mastihuba et al. 2002). Protein estimation was carried out using Bradford reagent and standard curve method (B6916; Sigma-aldrich, USA). Finally, specific activity (mU mg −1 ) of Fae was calculated for intracellular and secretory protein fractions from both control and rice-extract-treated samples.
Similarly, FAEs gene expression profile during different stages of infection (in vivo) was studied using a method, with modifications, reported earlier (Skamnioti and Gurr 2007). Briefly, a 15-20 µL WT conidial suspension (~ 10 5 conidia/mL; containing 0.05% gelatin) was drop-inoculated on the surface-sterilized 2-3-week-old barley leaf blades placed on to kinetin-agar plates, followed by incubation under dark (8 h) and light (14 h) cycles at 25 °C. Samples for RNA extraction were collected by excising inoculated portion of the leaf blades at different time-points, viz., 12-, 24-, 48-, 72-, and 96-h post-inoculation (hpi) along with 3-day-old vegetative mycelia, grown in liquid CM, as a control condition for qRT-PCR analysis. A sample from mockinoculation, i.e., leaves inoculated with only 0.05% gelatin solution, was used as a negative control for any non-specific amplification during qRT-PCR.
Total RNA was extracted from the samples collected for each of the above-mentioned conditions/time-points using TRIzol ® reagent (Invitrogen, USA), as per the manufacturer's instructions. A total of 2 µg of total RNA each were used for the first-strand cDNA synthesis using Oligo-(dT) 18 primers (Sigma, India) and M-MuLV reverse transcriptase (New England BioLabs, USA). The first-strand cDNA was then subjected to qRT-PCR analysis using a Power SYBR ® Green PCR Master Mix (Applied Biosystems, USA), performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems, USA) according to the manufacturer's instructions. Optimized thermal cycling conditions for qRT-PCR were as follows: initial denaturation step at 95 °C for 15 min followed by 40 cycling reactions each at 95 °C (denaturation step) for 15 s and 66 °C (annealing and extension step) for 45 s. Transcript levels in each of the test conditions were calculated by 2 −ΔΔCt method (Livak method;Livak and Schmittgen, 2001) relative to that of the vegetative mycelia grown in liquid MM or CM, normalized against β-tubulin (MGG_00604) transcript levels as an endogenous (loading) control. Primers used for qRT-PCR analysis are listed in the Supplementary Table S3.
Generation of FAE1 deletion mutant and genetic complementation of fae1Δ strain FAE1 gene (Gene ID: MGG_08737) deletion construct was made by double-joint PCR approach (Yu et al. 2004) using hygromycin phosphotransferase (HPT) gene as a selectable marker, followed by targeted gene replacement in WT M. oryzae via homologous recombination ( Supplementary Fig.  S1A). Here, ~ 1 kb each of 5′ and 3′-untranslated regions (UTR) of FAE1 gene were amplified from WT genomic DNA and fused with HPT gene cassette, using a recombinant PCR, followed by another round of PCR with nested primers to get a specific amplification product (Supplementary Table S4). This recombinant PCR product was further purified by Na-acetate/ethanol precipitation and used for polyethylene glycol (PEG)-mediated fungal transformation (Prakash et al. 2016), using WT M. oryzae protoplasts. Fungal transformants were selected on YEG agar plates containing 200 µg/mL hygromycin. Transformants growing on the selection medium were further screened by locusspecific PCR, where only one out of a total 72 transformants showed the desired amplified PCR product ( Supplementary  Fig. S1B). This transformant was further analysed by RT-PCR, wherein, as expected, no transcript was detected when compared with the WT or an ectopic transformant (Supplementary Fig. S1C).
Next, the selected transformant was confirmed for sitespecific integration (replacement of the FAE1 ORF with the selectable marker gene) by Southern blot hybridization. Genomic DNA was extracted from the WT and fae1Δ strains and further subjected to restriction enzyme digestion with PstI. The HPT gene, used as a probe, was labeled and DNA detection was performed using Alkphos Direct labeling and detection kit (GE Healthcare Ltd., UK) following the manufacturer's instructions. Southern blot hybridization analysis confirmed replacement of the FAE1 ORF in the mutant with a single copy of HPT gene cassette ( Supplementary Fig.  S1D).

Whole-plant infection and in vitro host-invasion assays
Whole-plant infection assay was carried out by spraying ~ 10 5 conidia/mL in 0.05% w/v gelatin onto ~ 4-weekold rice plants, followed by incubation at 28-30 °C under humid conditions, initially for 24 h in the dark and then for 4-6 days under 14 h light and 10 h dark cycles. Development of disease symptoms was monitored regularly during the entire incubation period and recorded at an appropriate time.
Host invasion by the fungal strains was checked by inoculating ~ 20 µL conidial suspension (~ 10 4 conidia/mL) on to the leaf sheaths obtained from 3-4-week-old host (rice, wheat or barley) plants, followed by incubation at room temperature (< 30 °C) under humid conditions. Host invasion was observed and quantified by scoring at least 100 appressoria each, using bright-field microscopy (Olympus BX51, Japan), for the development of invasive hypha in the plant cell underneath, at ~ 48 or 96 hpi. To check the effect of exogenous supply of glucose (2%, 1.5%, 1% or 0.5% w/v), ferulic acid (100 mM, 50 mM, or 10 mM), or their combinations (1% glu + 10 mM FA or 0.5% glu + 10 mM FA) or reduced glutathione (GSH, 20 mM) on host invasion by the blast fungus, the compound was added at 22 hpi to the rice leaf sheaths, already inoculated with conidia, and the resultant invasive hyphal growth was observed at ~ 48 hpi.

Statistical analyses
Quantitative analyses for fungal Fae enzyme activity, transcript levels, vegetative growth, conidiation, appressorial development, and host-invasion ability were carried out with three independent sets and the results are reported as mean ± standard deviation of mean (SDM). Statistical significance was determined using two-tailed t-test; and P values less than 0.05 (i.e. < 5%) were considered as statistically significant. P < 0.05 is denoted as * and likewise P < 0.01 as ** and P < 0.001 as ***.

Identification and in silico analysis of feruloyl esterase (FAE) gene(s) in M. oryzae
We performed a protein BLAST analysis using the known type B Fae sequences from Aspergillus oryzae (AoFaeB; PDB: 3WMT_B) and Neurospora crassa (NcFaeB; Gen-Bank: AJ293029) as a query against M. oryzae 70-15 reference genome database. We found in M. oryzae genome a total of nine putative feruloyl esterases that showed homology with either AoFaeB or NcFaeB. While MGG_08737 and MGG_07294 showed highest (47% and 75%) identity to AoFaeB and NcFaeB, respectively (Supplementary  Table S2), MGG_03771 showed similarity to both the reference sequences. Feruloyl esterases belong to the α/βhydrolase-fold superfamily and catalyze substrate hydrolysis following the mechanism of serine proteases with a conserved GXSXG motif and Ser-His-Asp/Glu catalytic triad (Dilokpimol et al. 2016). Our multiple sequence alignment of all nine putative Fae in M. oryzae showed the presence of the conserved GXSXG motif (Fig. 1A).
Next, we performed an HMM-based search for the presence of Tannase (Pfam ID: PF07519.13) or Esterase_PHB (Pfam ID: PF10503.13) domain, another characteristic of Fae, within the protein sequences of 26 representative fungi, including M. oryzae (70-15), belonging to nine different taxonomic classes and fifteen orders. We found in these fungi a total of 215 putative Fae protein sequences, of which 149 showed the presence of Tannase domain, whereas the remaining 66 protein sequences contained an Esterase_PHB domain (Supplementary Table S1). Here, the M. oryzae genome, unlike in the aforementioned GXSXG motif-based analysis, showed 12 putative Fae sequences, wherein nine (MGG_03771, MGG_03502, MGG_05366, MGG_02261, MGG_05592, MGG_08737, MGG_09404, MGG_09677, and MGG_09732) contained Tannase and the rest three (MGG_03746, MGG_07294, and MGG_10618) had Ester-ase_PHB domain. Interestingly, majority of the phytopathogenic fungi such as Fusarium spp., V. mali, Magnaporthe spp., Colletotrichum graminicola, and Zymoseptoria tritici showed more than one, Tannase domain containing, highly variable Fae sequences. However, Ustilago maydis, a basiodiomycete causal agent of corn smut disease, showed only two Esterase_PHB domain containing Fae sequences.
Intriguingly, the non-pathogenic filamentous fungi such as N. crassa, and particularly Aspergillus spp., also showed more than one putative Fae in their genomes, whereas fission yeast (S. pombe) or budding yeast (S. cerevisiae) did not show any Fae sequence.
Furthermore, we analysed the phylogenetic relationship of all the putative Fae, containing Tannase domain, in the aforementioned fungal species. The phylogenetic tree showed that Fae sequences had a significant genetic diversity and a discontinuous distribution pattern even among multiple Fae of the same species (Fig. 1B). All the clades, except for clade E and F, contained Fae sequences from species belonging to more than one taxonomic class. Whereas Fae sequences from clades E and F were found to be unique to class Sordariomycetes (Fig. 1B). Furthermore, all, except one, Fae sequences in clade E comprised of fungi predominantly belonging to the order Magnaporthales. Importantly, four out of nine putative Fae from M. oryzae (viz., MGG_02261, MGG_03502, MGG_05366, and MGG_05592), along with that from N. crassa, were grouped in clade E. The remaining five M. oryzae Fae orthologs were found to be grouped together with that from a distantly related genera like Fusarium, Colletotrichum and Aspergillus (clades B, C, G and I) as compared to their absence in a taxonomically closely related species N. crassa; suggesting a likely loss/reduction of Fae genes in the nonphytopathogenic fungus N. crassa. Interestingly, out of the aforementioned nine Tannase domain containing M. oryzae Fae, MGG_08737 (depicted as red star in Fig. 1B) was found to be highly diverged.
We then performed, using MEGA tool, a phylogenetic analysis of combined Fae sequences from host-specific M. oryzae strains, isolated from various cereal crops such as rice, wheat, and millet, and M. grisea as an outgroup ( Fig. 1C and Supplementary Fig. S2). Interestingly, our analysis showed that three M. oryzae Fae sequences, namely MGG_09404, MGG_09732, and MGG_08737, diverged likely in a host-specific manner ( Fig. 1C and Supplementary Fig. S2).

M. oryzae Fae are induced in the presence of host extract and are secretory in nature
We presumed that M. oryzae Fae would be required to function outside the cell; and hence studied whether these fungal enzymes were secretory in nature. First, we checked the presence or absence of a secretory signal peptide in the amino acid sequences of the aforementioned nine Fae using Fig. 1 Identification and phylogenetic analysis of putative fungal Fae . A Multiple sequence alignment of putative Fae sequences from M. oryzae, A. oryzae, and N. crassa. The conserved GXSXG motif is marked with a red box. B Phylogenetic analysis of Fae sequences from 18 fungal species representing four classes. The mid-point rooted phylogenetic tree is based on maximum-likelihood methods, with assessment of branch support by 1000 bootstrap replicates. Tree branches are color-coded according to the specific fungal species. The colors in the outer, middle, and inner circular strips represent taxonomy, for each branch, with respect to fungal class, order, and species, respectively. Bootstrap values are indicated as grey triangles, sized according to the values. Letters in uppercase from A to I denote the major clades of the tree. Stars denote Fae proteins from M. oryzae; red colored star marks the MGG_08737 gene used in the present study. Stars in clockwise direction, corresponding to leaf labels B41, C2, E5, E11, E15, E21, G7, I9, and I21 denote putative SignalP program. We found that a conventional secretory signal peptide was present in seven out of nine Fae in M. oryzae (Supplementary Table S2). To validate this observation, two putative FAE genes were randomly selected and subjected to the Yeast Secretion Trap (YST) system as described earlier ( Fig. 2A; Lee et al. 2006). The two FAE genes (MGG_05529 and MGG_07294) were then individually cloned in-frame, in the vector pYST2, with the SUC2 invertase gene without its native signal peptide, and transferred individually to the budding yeast (S. cerevisiae) strain DBYα2445, which does not carry its native SUC2 invertase gene. Thus, neither the untransformed yeast strain nor a transformant with the backbone vector would be able to grow on selection medium-containing sucrose as the sole carbon source. However, the yeast transformant expressing the Suc2 fused with Fae would be able to utilize sucrose in the selection medium, only if a signal peptide on Fae aids in secretion of the invertase. Indeed, the yeast transformants harboring the plasmid with Suc2 fused to either of the two FAE genes grew on sucrose agar, confirming the presence of a signal peptide and thereby the secretory nature of Fae enzymes in M. oryzae (Fig. 2B).
Next, to check whether the host tissue had any effect on M. oryzae Fae activity, the WT fungal culture was grown in YEG broth with or without crude rice leaf extract. Both, fungal biomass and culture supernatant, were collected separately and subjected to the in vitro enzyme activity assay. We found that the total enzyme activity was significantly higher (166 ± 3.33 mU mg −1 ; P < 0.001) in the extracellular (secretory) fraction of the culture grown in the presence of rice leaf extract, when compared with that (29 ± 0.69 mU mg −1 ) without the host extract (Fig. 2C). Importantly, the total enzyme activity in the intracellular fraction was less and remained largely unchanged even in the presence of the host leaf extract (Fig. 2C). These results indicate that most M. oryzae Fae are secretory and that their expression is induced by the host-derived factors suggesting a likely role for the CWDE in blast fungal pathogenesis.

M. oryzae Fae express differentially during pathogenesis
Given that the CWDEs are expressed under tight regulation (Zheng et al. 2009) and that M. oryzae Fae secretion was induced in the presence of rice leaf extract, we studied accumulation of nine FAE transcripts in response to individual host cell wall components. Considering the relatively higher turnover of the plant cell wall components during infection, we grew the vegetative culture in media containing-(1) Ferulic acid, (2) Pectin, (3) Xylan, (4) N-acetyl glucosamine (NAG; fungal cell wall component), (5) Cutin monomer (inducer of appressorial development), (6) Nitrogen starvation (mimic of pathogenic development), or (7) Glucose (control condition). Intriguingly, while there was no obvious pattern in accumulation of any particular transcript in response to the plant cell wall components, majority of the FAEs showed a ≥ twofold increase in expression in the presence of NAG (Fig. 3A). Whereas, those of almost all the FAEs were significantly lowered, either due to N 2 starvation or in the presence of xylan (Fig. 3A). Our findings suggest that the expression of M. oryzae FAE could be induced by likely activity of plant or fungal chitinase during the host-pathogen interaction.
Next, we studied the expression of FAEs during both pre-and post-host-invasion. The WT conidia were inoculated on detached barley leaves and samples were harvested at specific time-points signifying different stages of blast fungal infection cycle-pre-invasive appressorial development (12 h), host penetration, and colonisation (24-48 h) followed by necrotrophic growth phase (48-96 h). The relative transcript levels of all the nine FAEs were estimated by qRT-PCR during aforementioned infection stages and compared with those from the vegetative mycelia grown in liquid complete medium. While, almost all the FAE genes showed differential upregulation at different phases of pathogenic life cycle, remarkably, MGG_08737, hereafter referred as FAE1, showed a significant increase in relative transcript levels (~ 300-fold) during pre-invasive appressorial development and host penetration stages (12 and 24 hpi) when compared to those of the other FAEs from the vegetative mycelia (Fig. 3B). The FAE1 transcript levels further increased (~ 470-fold) during the subsequent host colonisation (48 hpi) and remained at elevated level (~ 293-fold) at 72 hpi, followed by a sharp decline (~ 27-fold) at 96 hpi (Fig. 3B). Importantly, the FAE1 transcript profile here is in accordance with the global transcriptome reported earlier in M. oryzae (Jeon et al. 2020). Our observations indicate that feruloyl esterases, particularly Fae1, have an important role during pathogenesis in the blast fungal pathogen.

M. oryzae Fae1 function is required specifically for host invasion
Given the significant upregulation of FAE1 during pre-and post-invasion, we generated a fae1Δ mutant to study its role, if any, in fungal development and pathogenesis. We first studied different phenotypic characteristics, such as vegetative mycelial growth and asexual (conidial) development, of the fae1Δ mutant. The vegetative growth of the fae1Δ mutant, after 10 dpi, was comparable to that of the WT, where colony size, morphology, and melanization on PA plates was similar in both the strains (Fig. 4A). While, the colony diameter of the WT was 7.37 ± 0.06 cm that of the fae1Δ was 7.17 ± 0.06 cm (Fig. 4B). Furthermore, total conidiation was determined by harvesting asexual conidia from vegetative culture on PA plates at 10 dpi. The fae1Δ produced 102.2 ± 12.2 × 10 2 conidia/cm 2 that was comparable to the 99.1 ± 7.4 × 10 2 conidia/cm 2 produced by the WT (Fig. 4C).
Given a significant increase in the FAE1 transcript levels also at the pre-invasive stage (12 hpi), we studied appressorial development in the fae1Δ mutant. Appressorial assay carried out on an artificial hydrophobic surface showed that the morphology of the fae1Δ appressoria was comparable to that of the WT appressoria observed at 24 hpi (Fig. 4D). We further quantified the appressorial development and found that the % appressoria formed was similar in both the WT (83.0 ± 1.5%) and fae1Δ mutant (81.7 ± 0.82%) (Fig. 4E). These results indicate that Fae1 does not play an important role in vegetative growth, asexual development, and hostindependent early pathogenic development in M. oryzae.
Next, we tested the pathogenicity of the fae1Δ mutant on the host, where rice or barley whole plants were sprayinoculated with conidia harvested from the WT, fae1Δ or fae1Δ/FAE1 strain and incubated under humid conditions for 5-6 days. Interestingly, the fae1Δ-inoculated plants did not show typical blast disease lesions, indicating that the mutant was significantly reduced in pathogenesis when compared with the WT or fae1Δ/FAE1 strain ( Fig. 5A and Supplementary Fig. S4A).
To further investigate the impaired pathogenesis in the fae1Δ strain, we studied the invasive growth, if any, of the mutant by microscopically observing the rice sheath inoculated with the WT or mutant. We found that most fae1Δ appressoria were unable to form visible primary invasive hyphae in rice sheath tissue (Fig. 5B). While 77.33 ± 5.41% and 56.88 ± 2.29% of the WT and fae1Δ/FAE1 appressoria, respectively, formed clearly visible invasive hyphae, only 3.28 ± 0.25% fae1Δ appressoria were able to invade and form primary invasive hyphae (P < 0.001; Fig. 5C). Furthermore, importantly, the primary invasive hyphae formed by the fae1Δ mutant were restricted in the first invaded host cell and failed to elaborate to the neighbouring cells (Fig. 5B). To rule out the possibility of delayed colonisation by the fae1Δ mutant, we checked the invasive growth at 96 hpi. Indeed, even after prolonged incubation, the fae1Δ mutant failed to colonise the plant tissue, as opposed to profuse invasive hyphal growth of the WT by then (Supplementary Fig. S3). Similar phenotypes were also observed for the fae1Δ mutant on other host plants such as barley and wheat (Supplementary Fig. S4B and S4C). Given that the Fae activity on the plant cell wall would release ferulic acid and polysaccharide molecules, we wondered whether exogenous supply of these compounds would enable the fae1Δ mutant in efficient/successful host tissue colonisation. First, we tested effect of ferulic acid in a drop-inoculation assay, where we found that the fae1Δ mutant successfully caused disease on the detached rice leaves, in the presence of exogenously added 100 mM compound (Fig. 5D). To find out whether ferulic acid supported the fae1Δ strain by reducing the oxidative stress, we assessed the ability of the mutant to cause disease in the presence of reduced glutathione (GSH), which is a known antioxidant. We found that the exogenously added 20 mM GSH failed to enable the fae1Δ mutant to invade the rice leaf tissue, when compared to the WT (Fig. 5D & E). This suggested that a different mechanism was responsible for the beneficial effect of ferulic acid accumulated/added at the host-pathogen interface. The alternate possibility was that the blast fungus had the ability to use ferulic acid as an energy source. Indeed, the vegetative mycelia of the WT M. oryzae could grow significantly on the basal medium with ferulic acid as the sole carbon source (Fig. 5F). With this, we decided to study the effect of different concentrations of both ferulic acid and glucose (considering the release of carbohydrates as well upon Fae activity) individually on host invasion by the fae1Δ mutant. Importantly, treatment with ferulic acid or glucose individually enabled an efficient invasive growth of the fae1Δ mutant, in a dose-dependent manner ( Fig. 6A; Supplementary Fig. S5A to S5C). While 2% glucose could significantly restore the host invasion (74.8 ± 9.8%) by the fae1Δ, comparable to that of the WT (75.4 ± 8.3%), other decreasing concentrations of glucose such as 1.5%, 1%, and 0.5% could also support the invasive growth of the fae1Δ to 50.2 ± 9.7%, 30.5 ± 5.5%, and 17.1 ± 3.2%, respectively ( Fig. 6A and B). Likewise, host invasion ability of the fae1Δ was improved upon exogenous addition of 100 mM (53.2 ± 5.9%) and 50 mM (29.3 ± 5.3%) ferulic acid (Fig. 6A and B). However, exogenous addition of 10 mM ferulic acid could show only a marginal increase in host invasion (7.1 ± 2.8%) by the fae1Δ mutant. A similar dose-dependent beneficial effect of glucose or ferulic acid could be observed on the infection ability of the fae1Δ on detached rice leaves ( Supplementary Fig. S5B). Furthermore, to check any synergistic effect of these two molecules, we performed a host invasion assay where the fae1Δ mutant was treated with two different combinations of glucose and ferulic acid (viz., 1% glu + 10 mM FA and 0.5% glu + 10 mM FA). We found that neither of these combinations, i.e., 1% glu + 10 mM FA and 0.5% glu + 10 mM FA, showed any remarkable improvement in host invasion by the fae1Δ, when compared to the WT or the mutant treated with glucose alone. Overall, these results suggest that the carbohydrates and/or ferulic acid released upon Fae activity likely act as an energy source for the blast fungus during host invasion.
Altogether, our results show that the Fae1-based likely plant cell wall degradation is required specifically for successful host invasion and colonisation during pathogenesis in M. oryzae. conditions. The cultures were grown in minimal medium with either 0.03% cutin monomers (1, 16-hexadecanediol), 0.03% ferulic acid, nitrogen starvation, 1% Pectin, 1% N-acetylglucosamine (NAG), or 1% Xylan, for 48 h before harvesting the biomass. The FAE transcript levels were estimated relative to those in the vegetative culture grown in minimal medium-containing 1% glucose as a control condition. The horizontal line corresponding to the fold change 1 represents normalized transcript levels for the control condition. B A bar chart depicting relative transcript levels of nine FAEs during different stages of pathogenic development. Samples were harvested at the time-points mentioned and the transcript levels were compared to those from vegetative mycelia as control condition. β-tubulin was used as an endogenous control in both (A) and (B). Data represent mean values ± s.d.m. from three independent biological replicates with technical triplicates each time

Discussion
Plant cell wall-degrading enzymes (CWDEs) play a pivotal role in virulence of phytopathogenic fungi. Feruloyl esterases belong to α/β-hydrolase-fold superfamily and catalyze substrate hydrolysis following the mechanism of serine proteases having a conserved motif GXSXG and a conserved Ser-His-Asp/Glu catalytic triad (Dilokpimol et al. 2016). Our in silico analysis of putative Fae in M. oryzae showed the presence of conserved GXSXG motif and were found to belong to α/β-hydrolase-fold superfamily.
Often, CWDEs are found as an expanded gene family in phytopathogenic fungi and are difficult to specifically characterize mainly due to tight transcriptional regulation and functional redundancy of the members of the gene family, i.e., loss of function of one gene is often compensated by the other genes in the family. Our HMM-based domain analysis across different fungal species showed a significant genetic diversity in the Fae sequences therein. Importantly, the absence of any putative Fae in budding and fission yeasts or human fungal pathogens (such as Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, and Mucor lusitanicus) suggests that the enzyme from other, especially phytopathogenic, fungal species are mainly associated with degradation of plant cell walls. Similarly, the presence of large number of Fae sequences in non-phythopathogenic filamentous fungi such as Aspergillus spp. and N. crassa is intriguing and studies on FAEs across different fungal genera could possibly shed some light on any evolutionary aspect of it. Indeed, our phylogenetic analysis revealed that the M. oryzae Fae1 (MGG_08737) has evolutionarily diverged more as compared to its paralogs and orthologs across different fungi. In M. oryzae, the previous studies were aimed at understanding the role of endo-xylanases (Nguyen et al. 2011) and cellulases (Vu et al. 2012) in fungal pathogenesis, by simultaneous silencing of multiple genes. Similarly, Cuti-nase2 (one out of the three putative Cutinases), which was significantly induced during host penetration stage, plays a role in full virulence in M. oryzae (Skamnioti and Gurr, 2007). In the present study, we investigated the role of one of the feruloyl esterases (Fae1) in pathogenicity of M. oryzae.
We found that the extracellular feruloyl esterase activity in the blast fungus was significantly increased in the presence of rice leaf extract. Possibly, FAEs were induced by the complex mixture of host factor(s) including the individual plant cell wall components. Thus, we studied the expression pattern of all the FAE genes under host-or pathogenicitymimic conditions. We found that FAE genes expressed differentially, with majority of them accumulating > 1.5 -fold higher, in response to individual plant cell wall components. While our observation is consistent with a previously reported similar upregulation of FAEs in Aspergillus niger (de Vries et al. 2002) and Fusarium graminearum (Balcerzak et al. 2012), it remains to be tested whether a combination of more than one host cell wall component would cause further upregulation in FAE expression. Intriguingly, majority of the FAEs were significantly upregulated in the presence of N-acetylglucosamine (NAG). A secreted M. oryzae chitinase (MoChia1), that binds to chitin to suppress plant immune response during infection (Yang et al. 2019), could likely digest free chitin to monomeric NAG, which in turn could be sensed by the blast fungus to express Fae. However, this hypothesis needs to be tested further.
A recent study on transcriptome profiling showed that MGG_08737 significantly upregulates at 18 hpi (161-fold), 27 hpi (82-fold), 36 hpi (130-fold), 45 hpi (147-fold), and 72 hpi (228-fold) during infection cycle of M. oryzae (Jeon et al. 2020). We found a similar pattern, where FAE1 was specifically upregulated during both pre-invasive appressorial development (12 and 24 hpi) and post-penetration host colonisation stages (48-72 hpi) of the infection cycle. Accordingly, loss of Fae1 function specifically impaired the ability of M. oryzae to invade and colonise rice, barley, and wheat tissue. Although our in silico analysis suggested that the Fae function could be attributed to specific hosts, the defect in the fae1Δ mutant could not be correlated to any of the host species used in this study. However, it remains to be studied whether fae1Δ has a distinct phenotype with any other cereal crops. Furthermore, very few appressoria (< 5%) that were able to form invasive hyphae in the mutant were defective in spreading to the adjacent host cells and were rather restricted to the first cell invaded, even after prolonged incubation (96 hpi). Our observations are in line with the hypothesis that Fae, and CWDEs in general, likely play an important role in cell-to-cell spread of the fungus within the host tissue, and subsequent necrotrophic growth phase (Zheng et al. 2009). We were intrigued by our observation that the deletion of just one FAE led to a significant defect in host invasion and that the presence of none other putative FAE could compensate for the loss of Fae1 function. Interestingly, our in silico analysis suggests that Fae1, among all the M. oryzae Fae, is highly diverged (Fig. 1B) and that there is no significant identity between any two Fae proteins in M. oryzae (Supplementary Fig. S6). This might possibly explain why the loss-of-function of Fae1 alone led to a significant phenotype in M. oryzae.
During appressorial development, the blast fungus derives energy by utilizing stored lipids via β-oxidation in the mitochondria and peroxisomes, generating acetyl-CoA, which is further distributed into the glyoxylate cycle and gluconeogenesis (Wang et al. 2007;Patkar et al. 2012). It is hypothesized that this metabolic process might be required to support the initial appressorial development and maturation, which ensures host penetration by the blast fungus, and the subsequent energy requirement during host tissue colonisation could be fulfilled by the host-derived nutrients (Fernandez and Wilson 2014). It is possible that the plant cell wall carbohydrates released after CWDEs activity could act as an energy source for the fungus at the host-pathogen interface and facilitate its entry and/or elaboration into the host. Feruloyl esterases in Aspergillus niger act synergistically, with other CWDEs like cellulases, xylanases, and pectinases, to degrade the complex plant cell wall carbohydrates (Faulds and Williamson, 1995). In M. oryzae, endo-xylanases (Nguyen et al. 2011) and cellulases (Vu et al. 2012) are shown to be important in host penetration and virulence of the blast fungus. Considering all this, we wondered whether carbohydrates released from CWDE-mediated plant cell wall digestion could act as an energy source for the blast fungus. Indeed, exogenous supply of glucose rescued the fae1Δ strain in a dose-dependent manner, indicating that Fae1 function is crucial for host invasion and colonisation during blast disease. A Whole-plant infection assay depicting blast disease outcome from the rice plants spray-inoculated with either the WT, fae1Δ, or fae1Δ/FAE1 conidia. The representative leaves were detached and photographed after 6 dpi. B Micrographs showing host invasion (~ 48 hpi) ability of either the WT, fae1Δ mutant or fae1Δ/FAE1. Images were taken at ~ 48 hpi. Asterisks mark appressoria, while arrowheads depict the invasive hyphae. Yellow asterisks mark the non-invading fae1Δ appressoria and the arrow depicts the invasive hypha restricted to the first invaded host (rice sheath) cell. Scale bar, 10 µm. C A bar chart depicting percentage appressoria invading rice sheath inoculated with either the WT, fae1Δ or fae1Δ/FAE1. Data represent mean ± s.d.m. from three independent experiments, with at least 100 appressoria each observed for quantification. ***, P < 0.001; ns, not significant. D Ferulic acid released by the Fae1 action is required during host invasion and colonisation by M. oryzae. Drop-inoculation assay showing infection ability (on detached rice leaves) of the WT, fae1Δ or fae1Δ supplemented with 100 mM ferulic acid (FA) or 20 mM reduced glutathione (GSH). Images were taken at 6 dpi. E Leaf sheath inoculation assay showing host invasion ability of the WT or fae1Δ supplemented with 20 mM GSH, which was added at 22 hpi. The results were observed at 48 hpi. White and yellow asterisks mark the invading and non-invading appressoria, respectively. Arrow depicts invasive hypha restricted to the first host cell invaded. Scale bar, 10 µm. F Ferulic acid likely acts as a nutrient source for M. oryzae. Vegetative growth of the WT M. oryzae on basal medium (BM) with or without 0.01% ferulic acid as the sole carbon source. The images were taken at 10 dpi ◂ the blast fungus is dependent on an exogenous source of energy at the time of host invasion. Interestingly, exogenous supply of ferulic acid, which is a product of Fae enzyme action, could also moderately support the invasive growth of the fae1Δ mutant in a dose-dependent manner. This is in accordance with a previous hypothesis that ferulic acid could possibly act as a weak or an alternative carbon source (Black and Dix 1976). Interestingly, a combination of glucose and ferulic acid showed a marginal yet synergistic response on reversal of the fae1Δ phenotype when compared to that by glucose alone (Fig. 6A and B). It is likely that either or both compounds released upon Fae activity serves as an energy source for the blast fungal pathogen during host invasion. We propose that the Fae in M. oryzae, like in A. niger, acts in a concerted manner on the esterified ferulic acid bridges in the plant cell wall, to allow endo-xylanases and cellulases to work on the carbohydrates therein, releasing constituent sugar molecules and ferulic acid, which could act as the energy source during host invasion (Fig. 7).
Feruloyl esterases have a wide range of applications in biofuel industry, food, cosmetic, and pharmaceutical industry, and also paper and pulp industry, all of which involve plant biomass degradation (Dilokpimol et al. 2016). It is often used in conjunction with other plant cell wall deconstructing enzymes. Most of the applied aspects of feruloyl esterases have been studied in Aspergillus spp. Although M. oryzae is a phytopathogenic fungus, one could explore the potential use of recombinantly expressed Fae1 in industrial applications, both in terms of enzyme activity and range of substrate specificity. Similarly, ferulic acid, the product of feruloyl esterase enzyme action, has a large application in food and pharmaceutical industry (Dilokpimol et al. 2016). Thus, use of an efficient feruloyl esterase (M. oryzae Fae1) for production of ferulic acid could also be explored. Moreover, it has been reported that Fae can also act on synthetic esterified substrates such as methyl ferulate, methyl sinapate, methyl p-coumarate, and methyl caffeate (Crepin et al. 2004). Therefore, it would be worth exploring whether or not Fae1, or feruloyl esterases in general, can also act on rutin complexed with glucose, thereby releasing quercetin, a plant flavanol with medicinal properties. This might also implicate another potential commercial application of Fae. Furthermore, given the involvement of Fae1 specifically in pathogenicity of M. oryzae, it could be considered as a potential target for developing an antifungal strategy.
Altogether, we show that the M. oryzae feruloyl esterase Fae1 plays a key role in pathogenesis, wherein the enzyme activity likely makes the alternative energy source available and supports the fungal growth during host invasion and colonisation. Fig. 6 Glucose or ferulic acid support host invasion in the fae1Δ in a dose-dependent manner. A Rice leaf sheath inoculation assay showing host invasion ability of the WT, fae1Δ or fae1Δ supplemented with varying concentrations of glucose (2%, 1.5%, 1%, and 0.5%), ferulic acid (100 mM, 50 mM, and 10 mM) or their combinations (1% glu + 10 mM FA and 0.5% glu + 10 mM FA), which were added at 22 hpi. The results were observed at 48 hpi. White and yellow asterisks mark the invading and non-invading appressoria, respectively. Arrows depict invasive hypha restricted to the first host cell invaded, while arrowheads represent invasive hyphae spreading to the neighbouring cells. Scale bar, 10 µm. B A bar chart depicting percentage appressoria invading rice sheath inoculated with either the WT, fae1Δ, or fae1Δ supplemented with different concentrations of glucose, ferulic acid, or their combinations. Data represent mean ± s.d.m. from three independent experiments, with at least 100 appressoria each observed for quantification. *P < 0.05; **P < 0.01; ns not significant ◂ Fig. 7 A proposed model of Fae1 function during pathogenesis in M. oryzae. Fae1, likely secreted along with other CWDEs, hydrolyses the plant cell wall to release ferulic acid and constituent carbohydrates during penetration of the first host cell and subsequent spread to the neighbouring cells. Released ferulic acid, the product of Fae enzyme action, and/or glucose, the breakdown product of cellulose, likely act as an energy source enabling successful host invasion and colonisation by the blast fungal pathogen